Receptor for trypsin and trypsin-like enzymes coupled to G proteins. Its function is mediated through the activation of several signaling pathways including phospholipase C (PLC), intracellular calcium, mitogen-activated protein kinase (MAPK), I-kappaB kinase/NF-kappaB and Rho. Can also be transactivated by cleaved F2R/PAR1. Involved in modulation of inflammatory responses and regulation of innate and adaptive immunity, and acts as a sensor for proteolytic enzymes generated during infection. Generally is promoting inflammation. Can signal synergistically with TLR4 and probably TLR2 in inflammatory responses and modulates TLR3 signaling. Has a protective role in establishing the endothelial barrier; the activity involves coagulation factor X. Proposed to have a bronchoprotective role in airway epithelium, but also shown to compromise the airway epithelial barrier by interrupting E-cadherin adhesion. Involved in the regulation of vascular tone; activation results in hypotension presumably mediated by vasodilation. Associates with a subset of G proteins alpha subunits such as G alpha-q, G alpha-11, G alpha-14, G alpha-12 and G alpha-13, but probably not with G(o) alpha, G(i) subunit alpha-1 and G(i) subunit alpha-2. However, according to PubMed21627585 can signal through G(i) subunit alpha. Believed to be a class B receptor which internalizes as a complex with arrestin and traffic with it to endosomal vesicles, presumably as desensitized receptor, for extended periods of time. Mediates inhibition of TNF-alpha stimulated JNK phosphorylation via coupling to G alpha-q/11; the function involves dissociation of RIPK1 and TRADD from TNFR1. Mediates phosphorylation of nuclear factor NF-kappa-B RELA subunit at 'Ser-536'; the function involves IKBKB and is predominantly independent of G proteins. Involved in cellular migration. Involved in cytoskeletal rearrangement and chemotaxis through beta-arrestin-promoted scaffolds; the function is independent of G alpha-q/11 and involves promotion of cofilin dephosphoryltaion and actin filament severing. Induces redistribution of COPS5 from the plasma membrane to the cytosol and activation of the JNK cascade is mediated by COPS5. Involved in the recruitment of leukocytes to the sites of inflammation and is the major PAR receptor capable of modulating eosinophil function such as proinflammatory cytokine secretion, superoxide production and degranulation. During inflammation promotes dendritic cell maturation, trafficking to the lymph nodes and subsequent T-cell activation. Involved in antimicrobial response of innate immnune cells; activation enhances phagocytosis of Gram-positive and killing of Gram-negative bacteria. Acts synergistically with interferon-gamma in enhancing antiviral responses. Implicated in a number of acute and chronic inflammatory diseases such as of the joints, lungs, brain, gastrointestinal tract, periodontium, skin, and vascular systems, and in autoimmune disorders.
Mammalian chitinase released by airway epithelia is thought to be an important mediator of disease manifestation in an experimental model of asthma. However, the intracellular signaling mechanisms engaged by exogenous chitinase in human airway epithelial cells are unknown. Here, we investigated the direct effects of exogenous chitinase from Streptomyces griseus on Ca(2+) signaling in human airway epithelial cells. Spectrofluorometry was used to measure intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-AM-loaded cells. S. griseus chitinase induced dose-dependent [Ca(2+)](i) increases in normal human bronchial epithelial cells and promoted [Ca(2+)](i) oscillations in H292 cells. Chitinase-induced [Ca(2+)](i) oscillations were independent of extracellular Ca(2+), suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum (ER) Ca(2+) with the ER Ca(2+) ATPase inhibitor, thapsigargin, chitinase-mediated [Ca(2+)](i) increases were abolished. Treatment with the phospholipase C (PLC) inhibitor U73122 or the 1, 4, 5-trisinositolphosphate (IP(3)) receptor inhibitor 2-APB attenuated chitinase-induced [Ca(2+)](i) increases. Desensitization of protease-activated receptor-2 (PAR-2) by repetitive agonist stimulation or siRNA-mediated PAR-2 knock-down revealed that chitinase-mediated [Ca(2+)](i) increases were exclusively mediated by PAR-2 activation. Finally, chitinase was found to cleave a model peptide representing the cleavage site of PAR-2 and enhanced IL-8 production. These results indicate that exogenous chitinase is a potent proteolytic activator of PAR-2 that can directly induce PLC/IP(3)-dependent Ca(2+) signaling in human airway epithelial cells.
J. Leukoc. Biol. 74, 60-68 (2003)[PubMed:12832443]
Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.
Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.
The airway epithelium is an important barrier between the environment and subepithelial tissues. The epithelium is also divided into functionally restricted apical and basolateral domains, and this restriction is dependent on the elements of the barrier. The protease-activated receptor-2 (PAR2) receptor is expressed in airway epithelium, and its activation initiates multiple effects including enhanced airway inflammation and reactivity. We hypothesized that activation of PAR2 would interrupt E-cadherin adhesion and compromise the airway epithelial barrier. The PAR2-activating peptide (PAR2-AP, SLIGRL) caused an immediate approximately 50% decrease in the transepithelial resistance of primary human airway epithelium that persisted for 6-10 min. The decrease in resistance was accompanied by an increase in mannitol flux across the epithelium and occurred in cystic fibrosis transmembrane conductance receptor (CFTR) epithelium pretreated with amiloride to block Na and Cl conductances, confirming that the decrease in resistance represented an increase in paracellular conductance. In parallel experiments, activation of PAR2 interrupted the adhesion of E-cadherin-expressing L cells and of primary airway epithelial cells to an immobilized E-cadherin extracellular domain, confirming the hypothesis that activation of PAR2 interrupts E-cadherin adhesion. Selective interruption of E-cadherin adhesion with antibody to E-cadherin decreased the transepithelial resistance of primary airway epithelium by >80%. Pretreatment of airway epithelium or the E-cadherin-expressing L cells with the long-acting beta-agonist salmeterol prevented PAR2 activation from interrupting E-cadherin adhesion and compromising the airway epithelial barrier. Activation of PAR2 interrupts E-cadherin adhesion and compromises the airway epithelial barrier.
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
J. Immunol. 167, 1014-1021 (2001)[PubMed:11441110]
The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca(2+) mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.
hPAR(2) (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR(2) by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR(2) that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys(361). We have demonstrated, using autoradiography, that Cys(361) is the primary palmitoylation site of hPAR(2). The hPAR(2)C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR(2)-expressing cell line. hPAR(2)C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR(2)C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR(2) possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR(2)C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR(2), whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR(2) expression, agonist sensitivity, desensitization and internalization.
Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.
J. Immunol. 180, 6903-6910 (2008)[PubMed:18453611]
Proteinase-activated receptor-2 (PAR(2)) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR(2) agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR(2) agonist to enhance IFN-gamma-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IkappaBalpha after costimulation of cells with PAR(2) agonist and IFN-gamma. Remarkably, coapplication of PAR(2) agonist and IFN-gamma also enhances the effects of IFN-gamma on IFN-gamma-inducible protein 10 kDa release, and CD64 and alphaVbeta3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR(2) activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR(2) agonists to enhance IFN-gamma-induced protective effects on human monocytes.
J. Immunol. 178, 5237-5244 (2007)[PubMed:17404307]
Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca(2+) mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca(2+)-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca(2+)-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2, and is a major source of PGE2, we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.
J. Cell Biol. 148, 1267-1281 (2000)[PubMed:10725339]
Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
Beta-arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), a Galpha(q/11)-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of beta-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Galpha(q)/Ca(2+) signaling and one through beta-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes beta-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Galpha(q)/Ca(2+) signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) Beta-arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that beta-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of beta-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Galpha(q/11) protein coupling.
J. Immunol. 167, 6615-6622 (2001)[PubMed:11714832]
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.
In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.
Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.
The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.
Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.
In this study we examined the potential for PAR(2) and TNFalpha to synergise at the level of MAP kinase signalling in PAR(2) expressing NCTC2544 cells. However, to our surprise we found that activation of PAR(2) by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFalpha stimulated IkappaBalpha loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR(4), however inhibition was reversed by pre-incubation with the novel PAR(2) antagonist K14585, suggesting that the effect is specific for PAR(2) activation. SLIGKV-OH was found to be more potent in inhibiting TNFalpha-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G(q/11) inhibitor YM25480 also reversed PAR(2) mediated inhibition. Activation of PAR(2) was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR(2) or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G(q/11), in mediating inhibition of TNFalpha-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.
Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils' responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils' innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.
Coagulation and inflammation are intimately linked and cellular signaling by coagulation proteases through protease-activated receptors (PARs) may affect pro- and anti-inflammatory responses. Permeability of the endothelial cell barrier at the blood-tissue interface plays a key role in inflammatory disorders such as sepsis. We have recently shown that PAR1 signaling by activated protein C or low concentrations of thrombin can enhance endothelial barrier integrity. In the present study, we analyzed effects of coagulation factor Xa (FXa), which is known to activate both endothelial cell PAR1 and PAR2, on monolayer integrity using a transformed human umbilical vein endothelial cell (HUVEC) line in a dual-chamber system. Preincubation with FXa potently reduced high-dose thrombin-mediated hyperpermeability and basal permeability. FXa was protective at concentrations of 5 nm or higher and proteolytic activity was required. Barrier protective FXa signaling was not affected by cleavage-blocking anti-PAR1 antibodies or by a PAR1 antagonist. Similarly, cleavage-blocking anti-PAR2 alone had no effect, but blocking both PAR1 and PAR2 inhibited barrier protection by FXa. Incubation of the cell layer with a PAR2-specific agonist peptide reduced thrombin-mediated hyperpermeability and basal permeability similar to FXa. In conclusion, not only PAR1, but also PAR2 can mediate barrier protection in endothelial cells and FXa can use either receptor to enhance barrier integrity. Although it is currently unknown whether PAR signaling by FXa has a physiological role, the results suggest a potential protective effect of FXa and other agonists of endothelial PAR2, which should be explored in models of local and systemic inflammation in vivo.
Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.
Autoantibodies to proteinase 3 (PR3) are involved in the pathogenesis of autoimmune-mediated vasculitis in Wegener granulomatosis (WG). To address the question how the autoantigen PR3 becomes a target of adaptive immunity, we investigated the effect of PR3 on immature dendritic cells (iDCs) in patients with WG, healthy blood donors, and patients with Crohn disease (CD), another granulomatous disease. PR3 induces phenotypic and functional maturation of a fraction of blood monocyte-derived iDCs. PR3-treated DCs express high levels of CD83, a DC-restricted marker of maturation, CD80 and CD86, and HLA-DR. Furthermore, the DCs become fully competent antigen-presenting cells and can induce stimulation of PR3-specific CD4(+) T cells, which produce IFN-gamma. PR3-maturated DCs derived from WG patients induce a higher IFN-gamma response of PR3-specific CD4(+) T cells compared with patients with CD and healthy controls. The maturation of DCs mediated through PR3 was inhibited by a serine protease inhibitor, by antibodies directed against the protease-activated receptor-2 (PAR-2), and by inhibition of phospholipase C, suggesting that the interactions of PR3 with PAR-2 are involved in the induction of DC maturation. Wegener autoantigen interacts with a "gateway" receptor (PAR-2) on iDCs in vitro triggering their maturation and licenses them for a T helper 1 (Th1)-type response potentially favoring granuloma formation in WG.
Combining with an extracellular signal and transmitting the signal across the membrane by activating an associated G-protein; promotes the exchange of GDP for GTP on the alpha subunit of a heterotrimeric G-protein complex.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
We recently characterized the proteinase-activated receptor (PAR)-2, a G protein-coupled receptor (GPCR), as the first cargo protein recognized by p24A. Here, we demonstrate that p24A binds to several other GPCRs, including PAR-1, the nucleotide receptors P2Y(1), P2Y(2), P2Y(4), and P2Y(11), as well as the μ-opioid receptor 1B. The acidic amino acid residues Glu and Asp at the second extracellular loop of GPCRs are essential for interaction with p24A. p23, another member of the p24 family, also interacts with GPCRs, similar to p24A. However, p23 shows a delayed dissociation from PAR-2 after activation of PAR-2, compared to the dissociation between PAR-2 and p24A. p24A and p23 arrest both P2Y(4) receptor and μ-opioid receptor 1B at the intracellular compartments, as observed for PAR-2. A comparable result was obtained when we studied primary rat astrocytes in culture. Over-expression of the N-terminal p24A fragment impairs PAR-2 resensitization in astrocytes that extends our findings to a native system. In summary, we demonstrate that p24A and p23 are specific cargo receptors of GPCRs and differentially control GPCR trafficking in the biosynthetic pathway, and thereby, p24A and p23 regulate GPCR signaling in astrocytes.
Evidence
2:
Inferred from Physical InteractionIntAct
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
Evidence
3:
Inferred from Physical InteractionIntAct
Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.
Evidence
4:
Inferred from Physical InteractionIntAct
Protease-activated receptor-2 (PAR-2), the second member of the G protein-coupled PAR family, is irreversibly activated by trypsin or tryptase and then targeted to lysosomes for degradation. Intracellular presynthesized receptors stored at the Golgi apparatus repopulate the cell surface after trypsin stimulation, thereby leading to rapid resensitization to trypsin signaling. However, the molecular mechanisms of the exocytic trafficking of PAR-2 from the Golgi apparatus to the plasma membrane remain largely unclear. Here we show that p24A, a type I transmembrane protein, which is a crucial constituent of the Golgi apparatus, associates with PAR-2 at the Golgi apparatus. The protein interaction occurs between the N-terminal region of p24A (residues 1-105; p24A-GL (GOLD domain with a small linker)) and the second extracellular loop of PAR-2. After receptor activation, PAR-2 dissociates from p24A. Importantly, we found that ADP-ribosylation factor 1 regulated the dissociation process and initiated PAR-2 trafficking to the plasma membrane. Conversely, overexpression of the fragment p24A-GL, but not other mutants containing the functional coiled-coil domain of p24A, arrested PAR-2 at the Golgi apparatus and inhibited receptor trafficking to the plasma membrane, which consequently prevented resensitization of PAR-2. These findings identify a new function of p24A as a regulator of signal-dependent trafficking that regulates the life cycle of PAR-2, Thus, we reveal a new molecular mechanism underlying resensitization of PAR-2.
Evidence
5:
Inferred from Physical InteractionIntAct
Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.
Biochem. J. 314 ( Pt 3), 1009-1016 (1996)[PubMed:8615752]
We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.
Interacting selectively and non-covalently with one or more specific sites on a receptor molecule, a macromolecule that undergoes combination with a hormone, neurotransmitter, drug or intracellular messenger to initiate a change in cell function.
Biochem. J. 314 ( Pt 3), 1009-1016 (1996)[PubMed:8615752]
We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.
Combining with thrombin to initiate a G-protein mediated change in cell activity. A G-protein is a signal transduction molecule that alternates between an inactive GDP-bound and an active GTP-bound state.
The sequential process in which the multiple coagulation factors of the blood interact, ultimately resulting in the formation of an insoluble fibrin clot; it may be divided into three stages: stage 1, the formation of intrinsic and extrinsic prothrombin converting principle; stage 2, the formation of thrombin; stage 3, the formation of stable fibrin polymers.
Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.
Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.
Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.
J. Immunol. 180, 6903-6910 (2008)[PubMed:18453611]
Proteinase-activated receptor-2 (PAR(2)) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR(2) agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR(2) agonist to enhance IFN-gamma-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IkappaBalpha after costimulation of cells with PAR(2) agonist and IFN-gamma. Remarkably, coapplication of PAR(2) agonist and IFN-gamma also enhances the effects of IFN-gamma on IFN-gamma-inducible protein 10 kDa release, and CD64 and alphaVbeta3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR(2) activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR(2) agonists to enhance IFN-gamma-induced protective effects on human monocytes.
Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.
J. Cell Biol. 148, 1267-1281 (2000)[PubMed:10725339]
Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
The establishment of a barrier between endothelial cell layers, such as those in the brain, lung or intestine, to exert specific and selective control over the passage of water and solutes, thus allowing formation and maintenance of compartments that differ in fluid and solute composition.
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
A series of molecular signals that proceeds with an activated receptor promoting the exchange of GDP for GTP on the alpha-subunit of an associated heterotrimeric G-protein complex. The GTP-bound activated alpha-G-protein then dissociates from the beta- and gamma-subunits to further transmit the signal within the cell. The pathway begins with receptor-ligand interaction, or for basal GPCR signaling the pathway begins with the receptor activating its G protein in the absence of an agonist, and ends with regulation of a downstream cellular process, e.g. transcription.
Eur. J. Biochem. 232, 84-89 (1995)[PubMed:7556175]
We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.
The immediate defensive reaction (by vertebrate tissue) to infection or injury caused by chemical or physical agents. The process is characterized by local vasodilation, extravasation of plasma into intercellular spaces and accumulation of white blood cells and macrophages.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
J. Immunol. 180, 6903-6910 (2008)[PubMed:18453611]
Proteinase-activated receptor-2 (PAR(2)) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR(2) agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR(2) agonist to enhance IFN-gamma-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IkappaBalpha after costimulation of cells with PAR(2) agonist and IFN-gamma. Remarkably, coapplication of PAR(2) agonist and IFN-gamma also enhances the effects of IFN-gamma on IFN-gamma-inducible protein 10 kDa release, and CD64 and alphaVbeta3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR(2) activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR(2) agonists to enhance IFN-gamma-induced protective effects on human monocytes.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
The process in which antigen-activated dendritic cells acquire the specialized features of a mature dendritic cell. Mature dendritic cells upregulate the surface expression of MHC molecules, chemokine receptors and adhesion molecules, and increase the number of dendrites (cytoplasmic protrusions) in preparation for migration to lymphoid organs where they present antigen to T cells.
Autoantibodies to proteinase 3 (PR3) are involved in the pathogenesis of autoimmune-mediated vasculitis in Wegener granulomatosis (WG). To address the question how the autoantigen PR3 becomes a target of adaptive immunity, we investigated the effect of PR3 on immature dendritic cells (iDCs) in patients with WG, healthy blood donors, and patients with Crohn disease (CD), another granulomatous disease. PR3 induces phenotypic and functional maturation of a fraction of blood monocyte-derived iDCs. PR3-treated DCs express high levels of CD83, a DC-restricted marker of maturation, CD80 and CD86, and HLA-DR. Furthermore, the DCs become fully competent antigen-presenting cells and can induce stimulation of PR3-specific CD4(+) T cells, which produce IFN-gamma. PR3-maturated DCs derived from WG patients induce a higher IFN-gamma response of PR3-specific CD4(+) T cells compared with patients with CD and healthy controls. The maturation of DCs mediated through PR3 was inhibited by a serine protease inhibitor, by antibodies directed against the protease-activated receptor-2 (PAR-2), and by inhibition of phospholipase C, suggesting that the interactions of PR3 with PAR-2 are involved in the induction of DC maturation. Wegener autoantigen interacts with a "gateway" receptor (PAR-2) on iDCs in vitro triggering their maturation and licenses them for a T helper 1 (Th1)-type response potentially favoring granuloma formation in WG.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
In this study we examined the potential for PAR(2) and TNFalpha to synergise at the level of MAP kinase signalling in PAR(2) expressing NCTC2544 cells. However, to our surprise we found that activation of PAR(2) by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFalpha stimulated IkappaBalpha loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR(4), however inhibition was reversed by pre-incubation with the novel PAR(2) antagonist K14585, suggesting that the effect is specific for PAR(2) activation. SLIGKV-OH was found to be more potent in inhibiting TNFalpha-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G(q/11) inhibitor YM25480 also reversed PAR(2) mediated inhibition. Activation of PAR(2) was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR(2) or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G(q/11), in mediating inhibition of TNFalpha-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
Negative regulation of tumor necrosis factor-mediated signaling pathwaydefinition[GO:0010804]
Any process that decreases the rate or extent of the tumor necrosis factor-mediated signaling pathway. The tumor necrosis factor-mediated signaling pathway is the series of molecular signals generated as a consequence of tumor necrosis factor binding to a cell surface receptor.
In this study we examined the potential for PAR(2) and TNFalpha to synergise at the level of MAP kinase signalling in PAR(2) expressing NCTC2544 cells. However, to our surprise we found that activation of PAR(2) by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFalpha stimulated IkappaBalpha loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR(4), however inhibition was reversed by pre-incubation with the novel PAR(2) antagonist K14585, suggesting that the effect is specific for PAR(2) activation. SLIGKV-OH was found to be more potent in inhibiting TNFalpha-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G(q/11) inhibitor YM25480 also reversed PAR(2) mediated inhibition. Activation of PAR(2) was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR(2) or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G(q/11), in mediating inhibition of TNFalpha-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
Beta-arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), a Galpha(q/11)-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of beta-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Galpha(q)/Ca(2+) signaling and one through beta-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes beta-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Galpha(q)/Ca(2+) signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) Beta-arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that beta-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of beta-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Galpha(q/11) protein coupling.
Any process that activates or increases the frequency, rate or extent of the directed movement of a motile cell or organism in response to a specific chemical concentration gradient.
ISSOrtholog Curator
Positive regulation of cytokine secretion involved in immune responsedefinition[GO:0002741]
Any process that activates or increases the frequency, rate, or extent of cytokine secretion contributing to an immune response.
J. Immunol. 167, 1014-1021 (2001)[PubMed:11441110]
The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca(2+) mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.
J. Immunol. 167, 6615-6622 (2001)[PubMed:11714832]
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.
J. Cell Biol. 148, 1267-1281 (2000)[PubMed:10725339]
Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
Any process that activates or increases the frequency, rate or extent of glomerular filtration. Glomerular filtration is the processs whereby blood is filtered by the glomerulus into the renal tubule.
In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.
Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.
J. Immunol. 178, 5237-5244 (2007)[PubMed:17404307]
Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca(2+) mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca(2+)-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca(2+)-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.
In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.
BACKGROUND: We have previously reported the potentiation of PDGF-BB-induced chemotaxis of fibroblasts, vascular smooth muscle cells, and endothelial cells by FVIIa. Here we studied the role of TF/FVIIa and the induced signaling pathways in regulation of chemotaxis of human monocytes, fibroblasts, and porcine aorta endothelial cells. METHODS AND RESULTS: Human monocytes were obtained by using Ficoll-Paque gradient and the MACS system (for highly purified population), fibroblasts and PAE cells have been characterized previously. Inhibitors of selected signaling intermediates were used, and the effect of TF/FVIIa on the migratory response of all cells to chemotactic agents was analyzed. The induced signaling was studied by immunoprecipitation and Western blotting. TF/FVIIa complex selectively enhanced PDGF-BB-induced chemotaxis in a Src-family, PLC, and PAR-2-dependent manner. Using PAE cells we identified c-Src and c-Yes as the Src-family members activated by TF/FVIIa. We report for the first time the PAR-2 and Src family-dependent transactivation of PDGFRbeta by TF/FVIIa involving phosphorylation of a subset of PDGFRbeta tyrosines. CONCLUSIONS: The described transactivation is a likely mechanism of TF/FVIIa-mediated regulation of PDGF-BB-induced chemotaxis. Similar behavior of 3 principally different cell types in our experimental setup may reflect a general function of TF in regulation of cell migration.
Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.
Any process that activates or increases the frequency, rate or extent of the internalization of bacteria, immune complexes and other particulate matter or of an apoptotic cell by phagocytosis.
Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.
Any process that activates or increases the frequency, rate or extent of the directed movement of a motile cell or organism towards a higher concentration in a concentration gradient of a specific chemical.
BACKGROUND: We have previously reported the potentiation of PDGF-BB-induced chemotaxis of fibroblasts, vascular smooth muscle cells, and endothelial cells by FVIIa. Here we studied the role of TF/FVIIa and the induced signaling pathways in regulation of chemotaxis of human monocytes, fibroblasts, and porcine aorta endothelial cells. METHODS AND RESULTS: Human monocytes were obtained by using Ficoll-Paque gradient and the MACS system (for highly purified population), fibroblasts and PAE cells have been characterized previously. Inhibitors of selected signaling intermediates were used, and the effect of TF/FVIIa on the migratory response of all cells to chemotactic agents was analyzed. The induced signaling was studied by immunoprecipitation and Western blotting. TF/FVIIa complex selectively enhanced PDGF-BB-induced chemotaxis in a Src-family, PLC, and PAR-2-dependent manner. Using PAE cells we identified c-Src and c-Yes as the Src-family members activated by TF/FVIIa. We report for the first time the PAR-2 and Src family-dependent transactivation of PDGFRbeta by TF/FVIIa involving phosphorylation of a subset of PDGFRbeta tyrosines. CONCLUSIONS: The described transactivation is a likely mechanism of TF/FVIIa-mediated regulation of PDGF-BB-induced chemotaxis. Similar behavior of 3 principally different cell types in our experimental setup may reflect a general function of TF in regulation of cell migration.
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
J. Immunol. 167, 6615-6622 (2001)[PubMed:11714832]
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.
Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.
Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.
BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.
J. Immunol. 180, 6903-6910 (2008)[PubMed:18453611]
Proteinase-activated receptor-2 (PAR(2)) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR(2) agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR(2) agonist to enhance IFN-gamma-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IkappaBalpha after costimulation of cells with PAR(2) agonist and IFN-gamma. Remarkably, coapplication of PAR(2) agonist and IFN-gamma also enhances the effects of IFN-gamma on IFN-gamma-inducible protein 10 kDa release, and CD64 and alphaVbeta3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR(2) activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR(2) agonists to enhance IFN-gamma-induced protective effects on human monocytes.
Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.
The change in morphology and behavior of a mature or immature T cell resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, leading to the initiation or perpetuation of an immune response.
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Pathways
According to KEGG, this protein belongs to the following pathways:
Protein involved in immunity, any immune system process that functions in the response of an organism to a potential internal or invasive threat. The vertebrate immune system is formed by the innate immune system (composed of phagocytes, complement, antimicrobial peptides, etc) and by the adaptive immune system which consists of T- and B- lymphocytes.
Protein involved in the localized protective response to tissue damage, microbial infection, or the presence of foreign matter. It is characterized by swelling, redness, heat and pain and involves a complex series of events including vascular changes and accumulation of blood cells, such as neutrophil leucocytes and mononuclear phagocytes, at the site of injury.
Protein involved in innate immunity, an inborn defense mechanism used by organisms to defend themselves against invasion by pathogens (bacteria, fungi, viruses, etc.). Initially discovered in insects which are devoid of an adaptive immune system and rely only on innate immune reactions for their defense, this immediate response accomplishes many activities including recognition and effector functions. Recognition is mediated by broad specificity, pattern recognition, receptors which recognize many related molecular structures (e.g. polysaccharides, polynucleotides) present in microorganisms but not found in the host. The innate responses include the release of antimicrobial peptides, production of cytokines, acute- phase proteins, complement. Although many different innate immune mechanisms are deployed for host defence, a unifying theme of innate immunity is the use of germline-encoded pattern recognition receptors for pathogens or damaged self components, such as the Toll-like receptors, nucleotide-binding domain leucine-rich repeat (LRR)- containing receptors, retinoic acid-inducible gene I-like RNA helicases and C-type lectin receptors.
Receptors which transduce extracellular signals across the cell membrane. At the external side they receive a ligand (a photon in case of opsins), and at the cytosolic side they activate a guanine nucleotide-binding (G) protein. These receptors are hydrophobic proteins that cross the membrane seven times.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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